Widely employed substrates for assaying NAGase include p-nitrophenyl N-acetyl-.beta.-D-glucosaminide as disclosed in Biochemical Preparations, 10: 118 (1963) and 4-methylumbelliferyl N-acetyl-.beta.-D-glucosaminide as disclosed in Clinica Chimica Acta, 69(1): 85-91 (1976). The former substrate is disadvantageous in that a blank test is required for each specimen and the procedure for assaying is complicated. The latter substrate requires special appliances, such as a fluorophotometer. Further, m-cresol sulfophthalyl N-acetyl-.beta.-D-glucosaminide is known as an improved substrate for NAGase which does not need a blank test as described in U.S. Pat. No. 4,433,139 corresponding to European Pat. No. 0060793. However, since the assay method using this substrate is an end point method wherein a reagent for stopping an enzymatic reaction is added to the system and a color thus developed under an alkaline condition is determined, the assay should be done in a two-reagent system and also takes time so that apparatuses for automatization are limited.